Inhibition of Parasitic Farnesyl Diphosphate Synthase: Structural and Thermodynamic Studies

Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans, and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. Development of bisphosphonates as anti-parasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. As part of my doctoral studies, I determined the X-ray crystallographic structures of complexes of the FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates, at resolutions of 1.8 Å, 1.9 Å and 2.3 Å. Two of the inhibitors, 1-(2-hydroxy-2,2-bis-phosphono-ethyl)-3-phenyl-pyridinium (300B) and 1-(2,2-bis-phosphono-ethyl)-3-butyl-pyridinium (476A), co-crystallize with the homoallylic substrate, isopentenyl diphosphate (IPP), and 3 Ca2+ ions. A third inhibitor 3-fluoro-l-(2-hydroxy-2,2-bis-phosphono-ethyI)-pyridinium (46I), was found to bind two Mg2+ ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of LmFPPS and human FPPS provides new information for the design of bisphosphonates that will be more specific for LmFPPS inhibition. The structure of the LmFPPS-46I homodimer shows that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. We propose that IPP binds first to the open site that then closes, opening the site on the other monomer that closes after binding the second IPP leading to the symmetric, fully occupied FPPS dimer observed in other structures. Linear 2-alkylaminoethyl-1,1-bisphosphonates are effective agents against proliferation of Trypanosoma cruzi--the etiologic agent of American trypanosomiasis (Chagas disease)-- exhibiting IC50 values in the nanomolar range against the parasites. This activity is associated with inhibition at the low nanomolar level of the T. cruzi farnesyl diphosphate synthase (TcFPPS). X-ray structures and thermodynamic data of the complexes TcFPPS with five compounds of this family show that the inhibitors bind to the allylic site of the enzyme with their alkyl chain occupying the cavity that binds the isoprenoid chain of the substrate. The compounds bind to TcFPPS with unfavorable enthalpy compensated by a favorable entropy that results from a delicate balance between two opposing effects: the loss of conformational entropy due to freezing of single bond rotations, and the favorable burial of the hydrophobic alkyl chains. The data suggest that introduction of strategically placed double bonds and methyl branches should increase affinity substantially